Pathogenic bacteria require iron for their survival and virulence. The opportunistic pathogen Pseudomonas aeruginosa has multiple mechanisms by which it can acquire iron, including ferric and ferrous iron uptake systems. However, within the host P. aeruginosa can adapt to utilize heme via the heme assimilation (has) and Pseudomonas heme utilization (phu) systems. We have recently shown the OM receptor PhuR has a unique His-Tyr coordination, which is an emerging motif in high affinity heme acquisition systems. 13C-heme isotopic labeling studies combined with bacterial genetics suggest the PhuR receptor is the high capacity uptake receptor, with the HasR receptor acting primarily as a sensor and regulator of heme utilization. Furthermore, we have shown the heme metabolite biliverdin IX? is a feedback regulator of the heme sensing system (has), as well as several virulence mechanisms including the pyochelin and Zn/Ni-pseudopaline uptake system, Type III secretion systems (ExoS and ExoT), and extracellular proteases (LasB). The goal of the proposal is to understand the regulation and molecular mechanism of heme acquisition in P. aeruginosa. Specifically, we will elucidate the heme-dependent regulatory elements controlling expression of the has system through transcriptional and translational fusion studies. Targeted transcriptional and post-transcriptional studies will be complimented by global analysis through transcriptomic and and proteomic analyses. We will further define the substrate specificity of the bis-His HasR and His-Tyr coordinated PhuR and their respective contributions to heme acquisition and regulation. Contributions of the Has and Phu systems to heme acquisition and virulence within the host will be tested in murine acute and chronic lung infection models. In addition, dual RNA-seq will be performed to simultaneously determine the P. aeruginosa and murine host response to infection. MALDI- MSI will be used in combination with quantitative LC-MS methods to determine the spatial distribution heme metabolites (BVIX isomers) and host-pathogen biomarkers in PAO1 and heme utilization mutants. Completion of the studies will provide a molecular basis for P. aeruginosa adaption to heme utilization in the context of the host-pathogen interaction.